The cDNAs for three human UDP-N-Acetylgalactosamine; Polypeptide N- Acetylgalactosaminyltransferases (GaINAc transferases) have recently been cloned and sequenced. Recombinant, functional forms of these GaINAc transferases have been expressed. A partial cDNA has been obtained for a putative fourth member of this class of enzymes. These enzymes, which catalyze the addition of N-acetylgalactosamine (GaINAc) by an O-glycosidic linkage to serine and threonine residues (the first step in mucin-type glycosylation) show distinct specificities for some substrates and common specificities for other substrates (albeit with distinct kinetics). One implication of this finding is that the differential expression of these enzymes by different cells and organs may lead to the glycosylation of different sites on protein that are O-glycosylated with GaINAc. The positions of attachment of O-linked GaINAc to several mucin and mucin-like cell adhesion molecules will be evaluated for each of the recombinant GaINAc transferases. The substrates to be used include tandem repeats from different mucins and from mucin-like proteins such as the p-selection ligand PSGL-1. The patterns of expression of the different GaINAc transferases T1, T2, and T3 will be determined for normal tissues and tumors derived from them. The contribution of structural features of selected peptide substrates will also be e valuated by CD, NMR, and Molecular Modeling. The model system to be used will be the in vitro glycosylation of eleven residue peptides by different recombinant GaINAc transferases. One peptide (AHGVTSAPDTR) was selected for these studies for several reasons. It is among the best of the substrate peptides we have tested, its relative kcat/Km values is among the best of all substrates tested and it is a very effective substrate with all three of the recombinant transferases. Our initial characterization of structural features of this peptide using both NMR analysis and molecular modeling showed the presence of relatively stable structures. The peptide contains one threonine residue that is glycosylated, and one that is not glycosylated. Thus, investigation of this peptide may give us insight into molecular parameters that determine whether or not residues can be glycosylated by the action of these enzymes. In addition, we will characterize the nature of disulfide bonds among cysteine residues that are conserved among all GaINAc transferases cloned to date, and we will perform site specific mutagenesis on selected cysteine residues that may play a role in the catalytic activity of these enzymes.